The sulfoxide oxidation product of methionine is a derivative which is frequently encountered in nature, as well as in synthetic peptide chemistry. The ease with which it forms makes it a common impurity in most methionine-containing peptides isolated from natural sources.
It is most advisable with purified peptides to avoid storage conditions in which sulfoxide formation is favored [Toennies, G., and Callan, T. P. (1939) J. Biol. Chem. 129, 481]. Once oxidized, the reduced nucleophilicity of the parent thioether renders the amino acid less susceptible to other modifications. This is the basis of its use in peptide synthesis [Iselin, B. (1961) Helv. Chim. Acta 44, 61]and protein modification studies [Tashjian, A., Ontjes, D., and Munson, P. L. (1964) Biochem. 3, 1175]. The propensity of methionine, once purified, to oxidize, in addition to the requirement of reversible protection in synthesis, has led many investigators to substitute it with alternative amino acids [Kempe, T., Chow, F., Peterson, S. M., Baker, P., Hays, W., Opperman, G., L'Italien, J. J., Long, G., and Paulson, B. (1986) Biotechnology 4, 565; Krstenansky, J. L., Trivedi, D., Johnson, D., and Hruby, V. (1986) J. Am. Chem. Soc. 108, 1696; and Schalch, D., Reisman, D., Emler, C., Humbel, R., Li, C. H., Peters, M., and Lau, E. (1984) Endocrinology 115, 2490].
Selective and non-destructive reduction of methionine sulfoxide-containing peptides has been a recurrent problem in peptide chemistry [Kessler, W., and Iselin, B. (1966) Helv. Chim. Acta 49, 1330]. Most frequently, reduction is achieved through the use of high concentrations of reducing agents such as dithiothreitol or .beta.-mercaptoethanol at elevated temperatures [Houghten, R. A., and Li, C. H. (1979) Anal. Biochem. 98, 36]. The reaction rate is slow, and the process usually yields incomplete reduction. An alternative means of reduction has been dimethyl sulfide or thioanisole promotion of oxygen exchange in aqueous HCl [Savige, W. E., and Fontana, A. (1977) Adv. in Enzymology 47, 453; Shechter, Y. (1986) J. Biol. Chem. 261, 66], anhydrous HF [Tam, J. P., Heath, W. F., and Merrifield, R. B. (1983) J. Am. Chem. Soc. 105, 6442], 1M trifluoromethanesulfonic acid-TFA [Fujii, N., Kuno, S., Otaka, A., Funakoshi, S., Takagi, K., and Yajami, H. (1985) Chem. Pharm. Bull., Jpn. 33, 4587]. In these cases, reduction is facile and a variable degree of disulfide integrity can be maintained. Of notable concern, however, are the potential secondary consequences of exposure to strong acids of this nature.
Haloacids have been recognized to catalyze the transfer of oxygen from a sulfoxide to a sulfide for some time [Scorrano, G. (1973) Acc. Chem. Res. 6, 132]. Savige and Fontana, supra, were the first to explicitly describe the reduction of free (i.e., not part of a protein) methionine sulfoxide using 12 N HCl and dimethyl sulfide. Successful application to an oxidized lysozyme derivative was also described with minimal assessment of secondary side-reactions. They concluded that "the reaction conditions are rather severe and require HCl of high concentrations." Most recently, Shechter, supra, reported similar reduction of methionine sulfoxide in proteins through the use of dimethyl sulfide and varying concentrations of HCl. He concludes that "at 4.4-10.7 M HC1, the reaction proceeds to completion, while at lower concentrations of HCl (i.e. 1.0 M), the extent of the reduction is retarded tremendously." High performance analysis for secondary modifications was not performed in either study.
Methionine sulfoxide reduction in anhydrous HF or 1M trifluoromethanesulfonic acid-TFA is believed to occur by a different mechanism than that which occurs in aqueous HCl. In these strong anhydrous acids, reduction is believed to be a function of the acidity of the mixture and is not dependent on the nucleophilicity of the acid anion. Efficient reduction has been reported with minimal levels of undesirable modifications. However, the caustic nature of these solvents requires special handling precautions which impose severe restrictions on their use.
This invention is directed to a facile and highly selective process for treating proteins and peptides having methionine sulfoxide residues to selectively reduce such residues to methionine residues. Specifically, the process of this invention effects reduction using substantially milder conditions than those imposed by prior art methods.